*2.7. RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR (qRT-PCR)*

qRT-PCR was performed essentially as described [43]. Total RNA was isolated from 5 × 10<sup>7</sup> parasites using the InviTrap spin cell RNA mini kit (STRATEC Molecular GmbH, Berlin, Germany) according to manufacturer's instructions. First strand cDNA synthesis was performed using a mix of oligo-dT and random primers (QuantiTect Reverse Transcription kit, Qiagen, Hilden, Germany) following the manufacturer's protocol. Real-time qPCR reactions were performed in a 20 µL-reaction mixture consisting of 1 µL of cDNA sample, 0.5 µM each gene-specific forward and reverse primers, and 1 × DyNAmo Color Flash SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primers used for amplification of the target and reference genes are listed in Table S1. Reactions were run on a Rotor-GeneTM RG 3000 Instrument (Corbett, Sydney, Australia) using the following thermal cycling conditions: an initial denaturation step at 95 ◦C for 7 min, followed by 35 cycles at 95 ◦C for 15 s, 69 ◦C for 20 s, and 71 ◦C for 30 s. After PCR amplification, a step at 95 ◦C for 1 min was included, followed by a melting curve analysis (67–95 ◦C, hold 60 s on the first step, hold 8 s on next steps). Data collection and analysis were performed with the Rotor-Gene real-time analysis software 6.1.81 (Corbett, Sydney, Australia). The normalised expression ratio was calculated using the 2 –∆∆Cq method [44].
